Hector A. Bergonia

A porphomethene inhibitor of uroporphyrinogen decarboxylase causes porphyria cutanea tarda

Porphyria cutanea tarda (PCT), the most common form of porphyria in humans, is due to reduced activity of uroporphyrinogen decarboxylase (URO-D) in the liver. Previous studies have demonstrated that protein levels of URO-D do not change when catalytic activity is reduced, suggesting that an inhibitor of URO-D is generated in hepatocytes. Here, we describe the identification and characterization of an inhibitor of URO-D in liver cytosolic extracts from two murine models of PCT: wild-type mice treated with iron, δ-aminolevulinic acid, and polychlorinated biphenyls; and mice with one null allele of Uro-d and two null alleles of the hemochromatosis gene (Uro-d+/−, Hfe−/−) that develop PCT with no treatments. In both models, we identified an inhibitor of recombinant human URO-D (rhURO-D). The inhibitor was characterized by solid-phase extraction, chromatography, UV-visible spectroscopy, and mass spectroscopy and proved to be uroporphomethene, a compound in which one bridge carbon in the uroporphyrinogen macrocycle is oxidized. We synthesized uroporphomethene by photooxidation of enzymatically generated uroporphyrinogen I or III. Both uroporphomethenes inhibited rhURO-D, but the III isomer porphomethene was a more potent inhibitor. Finally, we detected an inhibitor of rhURO-D in cytosolic extracts of liver biopsy samples of patients with PCT. These studies define the mechanism underlying clinical expression of the PCT phenotype, namely oxidation of uroporphyrinogen to uroporphomethene, a competitive inhibitor of URO-D. The oxidation reaction is iron-dependent.

Crystal structure of human uroporphyrinogen III synthase.

Uroporphyrinogen III synthase, U3S, the fourth enzyme in the porphyrin biosynthetic pathway, catalyzes cyclization of the linear tetrapyrrole, hydroxymethylbilane, to the macrocyclic uroporphyrino gen III, which is used in several different pathways to form heme, siroheme, chlorophyll, F430 and vitamin B12. U3S activity is essential in all organisms, and decreased activity in humans leads to the autosomal recessive disorder congenital erythropoetic porphyria. We have determined the crystal structure of recombinant human U3S at 1.85 Å resolution. The protein folds into two α/β domains connected by a β‐ladder. The active site appears to be located between the domains, and variations in relative domain positions observed between crystallographically independent molecules indicates the presence of flexibility that may be important in the catalytic cycle. Possible mechanisms of catalysis were probed by mutating each of the four invariant residues in the protein that have titratable side chains. Additionally, six other highly conserved and titratable side chains were also mutated. In no case, however, did one of these mutations abolish enzyme activity, suggesting that the mechanism does not require acid/base catalysis.

Reduction of porphyrins to porphyrinogens with palladium on carbon

Porphyrinogens serve as substrates for three heme biosynthetic enzymes. Porphyrinogens are highly unstable and must be generated as an integral part of enzyme assays. Methods commonly employed to generate porphyrinogens include chemical reduction using sodium amalgam or sodium borohydride and enzymatic generation from porphobilinogen. Chemical reduction yields porphyrinogens in highly alkaline solutions with high ionic strength, whereas enzymatic generation requires purified enzymes, deproteination, and complete buffer replacement. This article describes an improved method for reducing porphyrins to porphyrinogens using palladium on carbon as a catalyst under hydrogen at ambient temperature and pressure in the dark. The palladium catalyst is removed by filtration, the filtrate is blown dry with an inert gas, and the dried porphyrinogen can be dissolved in a buffer compatible with biological studies.

Erythropoietin signaling regulates heme biosynthesis

Heme is required for survival of all cells, and in most eukaryotes, is produced through a series of eight enzymatic reactions. Although heme production is critical for many cellular processes, how it is coupled to cellular differentiation is unknown. Here, using zebrafish, murine, and human models, we show that erythropoietin (EPO) signaling, together with the GATA1 transcriptional target, AKAP10, regulates heme biosynthesis during erythropoiesis at the outer mitochondrial membrane. This integrated pathway culminates with the direct phosphorylation of the crucial heme biosynthetic enzyme, ferrochelatase (FECH) by protein kinase A (PKA). Biochemical, pharmacological, and genetic inhibition of this signaling pathway result in a block in hemoglobin production and concomitant intracellular accumulation of protoporphyrin intermediates. Broadly, our results implicate aberrant PKA signaling in the pathogenesis of hematologic diseases. We propose a unifying model in which the erythroid transcriptional program works in concert with post-translational mechanisms to regulate heme metabolism during normal development. DOI: http://dx.doi.org/10.7554/eLife.24767.001

UROPORPHYRIA IN THE Cyp1a2−/− MOUSE

Cytochrome P4501A2 (Cyp1a2) is important in the development of uroporphyria in mice, a model of porphyria cutanea tarda in humans. Heretofore, mice homozygous for the Cyp1a2-/- mutation do not develop uroporphyria with treatment regimens that result in uroporphyria in wild-type mice. Here we report uroporphyria development in Cyp1a2-/- mice additionally null for both alleles of the hemochromatosis (Hfe) gene and heterozygous for deletion of the uroporphyrinogen decarboxylase (Urod) gene (genotype: Cyp1a2-/-;Hfe-/-;Urod+/-), demonstrating that upon adding porphyria-predisposing genetic manipulations, Cyp1a2 is not essential. Cyp1a2-/-;Hfe-/-;Urod+/- mice were treated with various combinations of an iron-enriched diet, parenteral iron-dextran, drinking water containing δ-aminolevulinic acid and intraperitoneal Aroclor 1254 (a polychlorinated biphenyl mixture) and analyzed for uroporphyrin accumulation. Animals fed an iron-enriched diet alone did not develop uroporphyria but uroporphyria developed with all treatments that included iron supplementation and δ-aminolevulinic acid, even with a regimen without Aroclor 1254. Hepatic porphyrin levels correlated with low UROD activity and high levels of an inhibitor of UROD but marked variability in the magnitude of the porphyric response was present in all treatment groups. Gene expression profiling revealed no major differences between genetically identical triple cross mice exhibiting high and low magnitude porphyric responses from iron-enriched diet and iron-dextran supplementation, and δ-aminolevulinic acid. Even though the variation in porphyric response did not parallel the hepatic iron concentration, the results are compatible with the presence of a Cyp1a2-independent, iron-dependent pathway for the generation of uroporphomethene, the UROD inhibitor required for the expression of uroporphyria in mice and PCT in humans.

Mutation in human CLPX elevates levels of δ-aminolevulinate synthase and protoporphyrin IX to promote erythropoietic protoporphyria

Significance Although heme synthesis is ubiquitous, specific regulatory mechanisms couple heme production to cellular demand and environmental conditions. The importance of these regulatory mechanisms is highlighted by clinical variability in porphyrias caused by loss-of-function mutations in heme synthesis enzymes. Heme synthesis is also controlled by the mitochondrial AAA+ unfoldase ClpX, which participates in both heme-dependent degradation of δ-aminolevulinate synthase (ALAS) and ALAS activation. This study reports a human familial mutation in CLPX that contributes to erythropoietic protoporphyria (EPP) by partially inactivating CLPX. Reduced CLPX activity increases ALAS post-translational stability, causing pathological accumulation of protoporphyrin IX (PPIX) in human patients. Our results thus identify an additional gene that promotes PPIX overproduction and EPP and highlight the complex gene network contributing to disorders of heme metabolism. Loss-of-function mutations in genes for heme biosynthetic enzymes can give rise to congenital porphyrias, eight forms of which have been described. The genetic penetrance of the porphyrias is clinically variable, underscoring the role of additional causative, contributing, and modifier genes. We previously discovered that the mitochondrial AAA+ unfoldase ClpX promotes heme biosynthesis by activation of δ-aminolevulinate synthase (ALAS), which catalyzes the first step of heme synthesis. CLPX has also been reported to mediate heme-induced turnover of ALAS. Here we report a dominant mutation in the ATPase active site of human CLPX, p.Gly298Asp, that results in pathological accumulation of the heme biosynthesis intermediate protoporphyrin IX (PPIX). Amassing of PPIX in erythroid cells promotes erythropoietic protoporphyria (EPP) in the affected family. The mutation in CLPX inactivates its ATPase activity, resulting in coassembly of mutant and WT protomers to form an enzyme with reduced activity. The presence of low-activity CLPX increases the posttranslational stability of ALAS, causing increased ALAS protein and ALA levels, leading to abnormal accumulation of PPIX. Our results thus identify an additional molecular mechanism underlying the development of EPP and further our understanding of the multiple mechanisms by which CLPX controls heme metabolism.

Reductions in the mitochondrial ABC transporter Abcb10 affect the transcriptional profile of heme biosynthesis genes

ATP-binding cassette subfamily B member 10 (Abcb10) is a mitochondrial ATP-binding cassette (ABC) transporter that complexes with mitoferrin1 and ferrochelatase to enhance heme biosynthesis in developing red blood cells. Reductions in Abcb10 levels have been shown to reduce mitoferrin1 protein levels and iron import into mitochondria, resulting in reduced heme biosynthesis. As an ABC transporter, Abcb10 binds and hydrolyzes ATP, but its transported substrate is unknown. Here, we determined that decreases in Abcb10 did not result in protoporphyrin IX accumulation in morphant-treated zebrafish embryos or in differentiated Abcb10-specific shRNA murine Friend erythroleukemia (MEL) cells in which Abcb10 was specifically silenced with shRNA. We also found that the ATPase activity of Abcb10 is necessary for hemoglobinization in MEL cells, suggesting that the substrate transported by Abcb10 is important in mediating increased heme biosynthesis during erythroid development. Inhibition of 5-aminolevulinic acid dehydratase (EC 4.2.1.24) with succinylacetone resulted in both 5-aminolevulinic acid (ALA) accumulation in control and Abcb10-specific shRNA MEL cells, demonstrating that reductions in Abcb10 do not affect ALA export from mitochondria and indicating that Abcb10 does not transport ALA. Abcb10 silencing resulted in an alteration in the heme biosynthesis transcriptional profile due to repression by the transcriptional regulator Bach1, which could be partially rescued by overexpression of Alas2 or Gata1, providing a mechanistic explanation for why Abcb10 shRNA MEL cells exhibit reduced hemoglobinization. In conclusion, our findings rule out that Abcb10 transports ALA and indicate that Abcb10s ATP-hydrolysis activity is critical for hemoglobinization and that the substrate transported by Abcb10 provides a signal that optimizes hemoglobinization.

A method for determining δ-aminolevulinic acid synthase activity in homogenized cells and tissues

OBJECTIVE In mammalian cells the rate-limiting step in heme biosynthesis is the formation of δ-aminolevulinic acid (ALA). The reaction intermediates, porphyrins and iron and the final product, heme can be highly cytotoxic if allowed to accumulate. The importance of maintaining the levels of metabolic intermediates and heme within a narrow range is apparent based on the complex homeostatic system(s) that have developed. Ultimately, determining the enzymatic activity of ALA synthase (ALAS) present in the mitochondria is highly beneficial to confirm the effects of the transcriptional, translational and post-translational events. The aim of this study was to develop a highly sensitive assay for ALAS that could be used on whole tissue or cellular homogenates. DESIGN AND METHODS A systematic approach was used to optimize steps in formation of ALA by ALAS. Reducing the signal to noise ratio for the assay was achieved by derivatizing the ALA formed into a fluorescent product that could be efficiently separated by ultra performance liquid chromatography (UPLC) from other derivatized primary amines. The stability of ALAS activity in whole tissue homogenate and cellular homogenate was determined after extended storage at -80 °C. CONCLUSIONS A method for assaying ALAS has been developed that can be used with tissue homogenates or cellular lysates. There is no need to purify mitochondria and radiolabeled substrates are not needed for this assay. General laboratory reagents can be used to prepare the samples. Standard UPLC chromatography will resolve the derivatized ALA peak. Samples of tissue homogenate can be stored for approximately one year without significant loss of enzymatic activity.

Bacteroides fragilis requires the ferrous-iron transporter FeoAB and the CobN-like proteins BtuS1 and BtuS2 for assimilation of iron released from heme

The intestinal commensal and opportunistic anaerobic pathogen Bacteroides fragilis has an essential requirement for both heme and free iron to support growth in extraintestinal infections. In the absence of free iron, B. fragilis can utilize heme as the sole source of iron. However, the mechanisms to remove iron from heme are not completely understood. In this study, we show that the inner membrane ferrous iron transporter ∆feoAB mutant strain is no longer able to grow with heme as the sole source of iron. Genetic complementation with the feoAB gene operon completely restored growth. Our data indicate that iron is removed from heme in the periplasmic space, and the released iron is transported by the FeoAB system. Interestingly, when B. fragilis utilizes iron from heme, it releases heme‐derived porphyrins by a dechelatase activity which is upregulated under low iron conditions. This is supported by the findings showing that formation of heme‐derived porphyrins in the ∆feoAB mutant and the parent strain increased 30‐fold and fivefold (respectively) under low iron conditions compared to iron replete conditions. Moreover, the btuS1 btuS2 double‐mutant strain (lacking the predicted periplasmic, membrane anchored CobN‐like proteins) also showed growth defect with heme as the sole source of iron, suggesting that BtuS1 and BtuS2 are involved in heme‐iron assimilation. Though the dechelatase mechanism remains uncharacterized, assays performed in bacterial crude extracts show that BtuS1 and BtuS2 affect the regulation of the dechelatase‐specific activities in an iron‐dependent manner. These findings suggest that the mechanism to extract iron from heme in Bacteroides requires a group of proteins, which spans the periplasmic space to make iron available for cellular functions.